Microbiol. Vol.

The

Thermostable

Deoxyribonuclease

Screening

Method

for

Enterotoxin

V.K.

D.R.

Dairy

Thermostable

DNase

of dairy

lococcus

aureus

production some

such

been

methods

Further,

are

the

since

of

many

During

and

viable

Staphylococcus

total

do

aureus

65

lococcal

count

extract

agar

number

of

alone

samples

of

dairies the

and

bacteria

tem

et was

al

(11).

set

up

petri

plates.

until

further

extract 4 hr

use.

was at

indication micrococcal

37

The by

of

The

of

was

extracted extract

of

in

reagents.

method not

be

for

very

the

reliable

enterotoxigenic of but

milk,

preformed

the

toxigenic

(9).

dried

their

Hence,

collected

the

total

number

nature

of of

thermostable for the

agar

(4)

from

local

bacterial

DNase.

the

boiled

the

pre•|

used

markets

count,

Tryptone

were

were well.

pink A

Tris

test

staphy•|

dextrose

for

The

yeast

determining

in

the

petri

curve 8.5).

plates

was

95•|mm

and

(5•|7

C)

DNase

incubated was

prepared After

sys•|

diameter

5 ƒÊl of

were wells

of

assay

a refrigerator

agar

the

procedure

DNase

in

in the

the

The

medium

around

(pH

by

min.

stored

made

standard

437

15

agar

were

zones

buffer

samples

for

DNA

plates

each

DNase. (Sigma)

for

blue

wells to

Al•|

expensive

treatment,

products

from was

millimeter

development

(10).

respectively.

agar

transferred

Staphy•|

available,

carried out. The and reliable criterion

thermostable

Toluidine

solidification,

milk

manufacture

the

of

enterotoxin

already

of

to

of con•|

materials.

milk

S•|110

crude

thermostable nuclease

examined

staphylococci,

pouring

Two

gently C.

and

were

staphylococcus and

After

milk

use

indicate

food

dried

traditional

the

number through

and

are

detected.

also

large

growth

considered

heat be

always

in

the the

in

such

tests are satisfactory

in

foods

reported

milk

can

not

the

and

been

a

poisoning

DNase

in

are

and

more

(5)

is now

to

by

food

between

involve

has

survive

may

presence

Deoxyribonuclease Tatini

not

staphylococci

and (1)

or

given

other confirmatory be considered a

of

cheese

staphylococci

destroyed

not

of

thermostable

staphylococci

are

commercial

in

Research

produced

enterotoxins

which

treatment

enterotoxigenic

from

test

staphylococci DNase

unless test can

A and

heat

as

consuming

negative

drastic

toxins

of

of

enterotoxigenic

enterotoxigenic

sence

time

coagulase

coagulase

the

product DNase

either

plasma

identification

detection

be

well

Rapid

6, 1977)

correlation

as

demonstrated

for

to

a

1978

B.•@RANGANATHAN

outbreaks

A close DNase

well

methods

reported

as

Immunol. 437•|441,

Products

October

causing

(13).

thermostable

has

though

and

(7),

Staphylococcal

Milk

publication,

been (12)

products

and

of

and

GHODEKAR,

for

has

staphylococci

sumption

Detection

Milk

Test

Bacteriology Division, National Dairy Institute, Karnal, India

(Received

enterotoxigenic

the

in

BATISH,

(DNase)

22

extraction

the using

for positive purified

from

the

438

V.K. BATISH

ET AL

NOTES

439

Table 2. Determination of thermostable deoxyribonuclease and type of enterotoxin in DNase positive samples of milk and milk products

+, Present; -,

Absent.

samples (2), the enterotoxins were detected by microslide gel double diffusion tests (3). Standard antisera of enterotoxins A, B and C (supplied by Dr. M.S. Bergdoll) were used in this study. Brain Heart Infusion broth cultures were used for the determinations of thermostable DNase (7) and enterotoxin (3) as well as plasma coagulase (8) production by the staphylococcal isolates. Among 65 samples examined in this study, thermostable DNase was detected in only five samples. Data in Tables 1 and 2 show that thermostable DNase could be detected in only one each out of 17 cheddar cheese, five raw milk, 11 baby food samples and in two out of 17 skim milk powder samples. Viable staphylococcal counts in these samples ranged from 50 x 101-/g(a skim milk powder sample) to 16 X 105/ml (a raw milk sample). Tatini et al (10) found thermostable DNase in all of cheese samples used in their study where the staphylococcal population was 1.5 X 106/g. The results of the present study show that low staphylococcal counts in dried milk do not have any relation to the presence of detectable DNase. On the other hand, a high staphylococcal count of 16 x 105 per ml in one of the raw milk samples is suggestive of the presence of thermostable DNase and in fact the sample contained as much as 26 dugDNase/100 ml. In the remaining four samples, the thermostable DNase content ranged from 8-19 ,ug per 20 g. Enterotoxins A or B or both were detected in samples of milk and milk products which also contained DNase (Table 2). The incidence of enterotoxin A was, however, higher (80%) than that of enterotoxin B. Data in Table 2 indicate that determination of the viable S. aureuspopulation in finished milk products (especially heat-processed products) is therefore of limited value as a means of ascertaining whether or not enterotoxins are present. Samples having low viable S. aureus counts which are not suggestive of a potential problem may contain detectable levels of thermostable DNase and enterotoxins. Similar findings were observed by Tatini et al (10) in butter, nonfat dry and dried malted milk samples. Out of 236 staphylococcal isolates examined in the current study, 29 were positive for presence of thermostable DNase, coagulase and enterotoxins, while six other coagulase positive isolates failed to produce either thermostable DNase or enterotoxins (Table 3). All thermostable DNase-producing isolates collected from different

440

V.K. BATISH Table

samples

3.

Production of thermostable deoxyribonuclease, by staphylococci isolated from milk and

produced

sample

enterotoxin

which Rayman

et

al

aureus.

results

will

In

the

light

thermostable

The consin,

in

the

subject

of

the

for

enterotoxins

authors

are

Madison,

respective

in

indebted

be

this

a

Dr.

the

to

of

reliable dairy

Bergdoll,

either In

negative et

al

obtain

S.

(6)

aureus

reported

a

produced

by

thermostable

DNase

S.

DNase

positive

cells

and

the

and

present

study

relatively

suggest

simple

that

screening

products.

Food

for

produce

DNase

from

findings

including

U.S.A.

powder

communication.

rapid,

M.S.

53706,

milk

producers.

Lachica heat•|stable

laboratory

separate

to

coagulase coagulase

production

a

foods

to

Wisconsin

in

and

a skim

failed were

However,

report,

can

from

enterotoxins

both.

certain

enterotoxin

above test

or

they

enterotoxin

of

isolates) B

demonstrated

initiated

coagulase and milk products

staphylococci

enterotoxins.

been

DNase

method

(9)

deficient form

some

or

although

coagulase,

have

mutants

(five A

that

produce

among

Studies

negative

those

enterotoxin,

Kato

could

except enterotoxin

reported

and

relationship

the

(8) or

Omori which

fair

either

DNase

strains

A

produced

thermostable contrast,

ET AL

Research

supplying

Institute,

The

staphylococcal

University

of Wis•|

enterotoxins

and

their

antisera.

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